Ultrafast time-resolved near- and mid-IR spectroscopy will be employed to probe the structure, dynamics, and function of ligand- binding heme proteins. Transient IR spectra of photolyzed myoglobin (MbCO, MbNO, and Mb mutants recorded with less than 200 fs time resolution and with high sensitivity will reveal the orientation of ligands bound to and dissociated from the heme, the nature of ligand motion within the protein, the influence of solvent on protein and ligand dynamics, and the functional role of highly conserved residues in the protein. The incisive information about ligand orientation and motion afforded by this experimental approach allows detailed testing of structure-function hypotheses and molecular dynamics simulations and thereby probes the origins of molecular recognition and control. The orientation of ligands bound to and dissociated from the heme of myoglobin (Mb) in solution will be determined using the technique of photoselection spectroscopy. This technique, which has been successfully applied to CO, will be used to determine the orientation of bound NO and O2 and the orientation of NO dissociated from the heme of Mb. The influence of crystal packing forces on both ligand and protein dynamics will be probed. In addition, the influence of the solvent and its viscosity on both ligand and protein dynamics will be determined. The functional role of highly conserved residues in the vicinity of the ligand binding site in Mb will be explored by femtosecond time- resolved IR studies of MbCO mutants. Time-resolved IR studies will be extended to hemoglobin (Hb), a tetrameric allosteric protein.
